The invention relates to improved epoxide hydrolases which can be isolated from bacteria of the genus Streptomyces, a novel process for the enzymatic separation of epoxide enantiomer mixtures, a novel detection method for epoxide hydrolase activity, a screening method for detecting epoxide hydrolase activity and the use of bacteria of the genus Streptomyces and the resultant epoxide hydrolases for enantioselective epoxide hydrolysis.
The increasing importance of enantiomerically pure compounds, especially in the pharmaceutical and agrochemical industries, requires reliable and economic access to optically active substances. To prepare enantiomerically pure diols and epoxides, a number of methods are available.
In asymmetric chemical synthesis of epoxides, synthesis starts from a prochiral compound. By using a chiral reagent, for example a chiral peracid, a chiral dioxirane or oxaziridine or a chiral borate, a chiral auxiliary or a chiral, metallic or nonmetallic catalyst, a chiral epoxide is formed. The best known pathway for synthesizing chiral epoxides is the Sharpless epoxidation of alkenols, for example allyl alcohols, with hydroperoxides in the presence of transition metal catalysts.
Preparation of enantiomerically pure diols by a biochemical pathway is also known. Microorganisms having epoxide hydrolase activity catalyze the regiospecific and enantiospecific hydrolysis of epoxides. They cleave the ether bond in epoxides, forming diols. Some bacterial strains have already been described which enable a broad selection of racemic epoxides to be hydrolyzed enantioselectively. However, the number of known epoxide hydrolases and their application to organic synthesis has been restricted to date. Known strains having epoxide hydrolase activity are, for example, Aspergillus niger LCP521, Bacillus sulfurescent ATCC 7159, Rhodococcus species NCIMB 11216 and others.
However, the known strains having epoxide hydrolase activity often have a restricted substrate spectrum and low reaction rates. Also, the enantioselectivities which can be achieved using these strains are frequently too low [Grogan, G., et al., FEMS Microbiology Lett. 141 (1996), 239-243; Kroutil, W., et al., Tetrahedron Lett. (1996), 8379-83821. The known strains are difficult to manipulate genetically and some are difficult to culture. Therefore, to date, only two epoxide hydrolases are available in recombinant form in E. coli [Corynebacterium sp. C12, (Misawa, E., et al., Eur. J. Biochem. 253 (1998), 173-183) and Agrobacterium radiobacter AD1 (Rink, R., et al., J. Biol. Chem. 272 (1997), 14650-14657)]. Even purification of the epoxide hydrolases obtained from the microorganisms has to date only been described for Rhodococcus species NCIMB 11216 [Faber, K., et al., Biotechnology Lett. 17 (1995), 893-898] and Norcardia EH 1 [Kroutil, W., et al J. Biotechnol. 61 (1998), 143-150]. This was highly complex in both cases. The enrichment of epoxide hydrolases from Corynebacterium sp. C12 is described by Misawa, E., et al., Eur. J. Biochem. 253, (1998) 173-183.
In addition, the search for novel epoxide hydrolase-containing microorganisms is made difficult owing to the fact that screening for novel epoxide-hydrolase-producing microorganisms, for example in collections of microbiological strains, has hitherto been highly time-consuming. This is due to the fact that, for screening, usually methods are used in which the individual batches must be worked up and analyzed individually by gas chromatography or liquid chromatography.
It is a first object of the invention, therefore, to provide novel epoxide hydrolases having an expanded substrate spectrum and/or improved reactivity and/or improved enantioselectivity. In addition, the novel epoxide hydrolases should be more readily accessible, in particular, because they can be isolated from nonpathogenic organisms which can be readily cultured, and, in addition, if appropriate are readily accessible to methods of molecular biology.
It is a second object of the invention to provide a method for the more rapid and simpler detection of epoxide hydrolase, which should also allow improved screening for epoxide-hydrolase-producing micro-organisms.
It is a third object of the invention to provide an improved biochemical process for separating epoxide enantiomer mixtures and thus an improved process for the enantioselective reaction of epoxides which permits a simpler route to the enantiomerically pure diols and/or epoxides.
It is a fourth object of the invention to provide novel epoxide-hydrolase-producing microorganisms.
We have found that the above first object is achieved, surprisingly, by providing epoxide hydrolases (E.C. 3.3.2.3) from microorganisms of the genus Streptomyces. Epoxide hydrolase activity has not previously been described in microorganisms of this genus.
The inventive epoxide hydrolases have at least one of the following advantageous properties, compared with previously known epoxide hydrolases:
improved enantioselectivity in the resolution of enantiomeric epoxides;
improved (expanded) substrate spectrum;
improved reactivity;
enhanced accessibility to methods of molecular biology;
improved biochemical accessibility because the microorganisms are easier to culture.
For the purposes of the invention, xe2x80x9cimproved enantio-selectivityxe2x80x9d is when, for substantially the same conversion rate, a higher enantiomeric excess is achievable.
For the purposes of the invention, an xe2x80x9cexpanded substrate spectrumxe2x80x9d is that racemic mixtures of a plurality of epoxides are converted.
For the purposes of the invention, an xe2x80x9cimproved reactivityxe2x80x9d is that the reaction takes place with a higher space-time yield.
The invention relates in particular to those epoxide hydrolases from Streptomyces that have at least one of the following properties:
a) hydrolytic epoxide cleavage of a styrene oxide, for example styrene oxide or a derivative thereof which is monosubstituted or polysubstituted on the phenyl ring or epoxide ring, such as in particular a styrene oxide which is monosubstituted in the meta or para position by nitro or halogen, in particular chlorine or bromine, and at least one further compound selected from the group consisting of ethyl 3-phenylglycidate, n-hexane-1,2-oxide, n-decane-1,2-oxide and indene oxide, which can be unsubstituted or monosubstituted or polysubstituted by substituents preferably in accordance with the above definition;
b) conversion of a racemate of styrene oxide with an enantioselectivity E greater than 2, for example xe2x89xa710, for instance from 10 to 100, to give (S)-phenyl-1,2-ethanol according to the reaction equation (A) given below, this conversion being able to be carried out using whole cells or a cell homogenate or an enriched or purified enzyme preparation, and preferably taking place in the presence of a cosolvent, for example from 5 to 10% (v/v) DMSO.
According to a preferred embodiment, the epoxide hydrolases provided are those which can be isolated from bacteria of the genus Streptomyces, in particular from the species S. griseus, S. thermovulgaris, S. antibioticus, S. arenae and S. fradiae, preferably from the strains Streptomyces griseus (DSM 40236 and DSM 13447), Streptomyces thermovulgaris (DSM 4044 and DSM 13448), Streptomyces arenae Txc3xc (DSM 40737 and DSM 12134) Streptomyces antibioticus Txc3xc4 (DSM 12925) or Streptomyces fradiae Txc3xc27 (DSM 12131).
Particular preference is given to the epoxide hydrolase which can be isolated from Streptomyces antibioticus Txc3xc4 (DSM 12925). This enzyme is characterized by its pronounced enantioselectivity and the conversion of (R/S)-styrene oxide (I) according to the following reaction equation (A)
Reaction equation (A): 
to (S)-phenyl-1,2-ethanediol (III), with the non-hydrolysis of (R)-styrene oxide (II). Thus this enzyme in a reaction medium containing 10% (v/v) DMSO as solubilizer, catalyzes the above conversion at an enantioselectivity of E=13 and an enantiomeric excess ee[%]=99 for (II) and ee[%]=14 for (III).
Isolation of the enzyme is described in more detail in the examples. Unless stated otherwise, the enzyme is enriched using standard biochemical methods, for example as described by T. G. Cooper in Biochemische Arbeitsmethoden [Biochemical methods], Verlag Walter de Gruyter, Berlin, N.Y., (1981). Suitable methods, for example, are purification methods such as precipitation, for example using ammonium sulfate, ion-exchange chromatography, gel chromatography, affinity chromatography, for example immunoaffinity chromatography, and isoelectric focusing, and combinations of these methods.
The invention also relates to a novel Streptomyces strain having the designation Streptomyces antibioticus Txc3xc4, deposited at the DSMZ under the Deposit Number DSM 12925 and variants and mutants of this strain.
The invention also relates to functional analogs of the inventively prepared enzymes, such as variants, alleles and mutants, that have epoxide hydrolase activity and, preferably, have at least one of the above-mentioned advantageous properties.
We have found that the above second object was achieved, surprisingly, by providing an optical method of detection for epoxide hydrolase, which comprises
a) incubating an analyte, for example a microorganism culture, in which epoxide hydrolase activity is suspected with an epoxide-containing substrate for the epoxide hydrolase under reaction conditions;
b) chemically reacting unreacted epoxide with 4-nitrobenzylpyridine (NBP), forming a pigment absorbing at 560 nm; and
c) analyzing the solution from step b) for decrease in pigment concentration, relative to an epoxide-hydrolase-free control solution.
The inventive detection method can be carried out qualitatively, for example as a spot test, or quantitatively. In the quantitative method, the relative decrease in pigment concentration is first determined quantitatively, for example photometrically by determining absorption at 560 nm, and the epoxide hydrolase activity in the analyte is determined therefrom.
Suitable analytes are in principle microorganisms per se, for example samples from a freshly taken culture of a bacterium, cell homogenates thereof or fractions of these cell homogenates after purification. Preferably, the test is carried out using whole cells or after digestion of the cells, for example using ultrasound or lysozyme.
For example, the method is applicable to detecting epoxide hydrolase in bacteria of the genus Streptomyces. 
Preferably, the detection method is carried out in such a manner that a sample is withdrawn from a freshly taken culture of the microorganism, this is disrupted, freed from cell fragments and an epoxide comprising a substrate for the enzyme to be tested is added and mixed. If required, the reaction conditions can be optimized in the solution by customary measures, for example by adding buffer, adjusting the reaction temperature and the like. Preferably, to improve the solubility of the epoxide in the aqueous reaction medium, a cosolvent is used. Suitable cosolvents are, for example, dimethyl sulfoxide (DMSO), dimethylformamide (DMF), ethanol or acetone. Optimum reaction conditions for epoxide hydrolases from Streptomyces comprise:
Reaction medium: Sodium phosphate buffer pH 8.0, 0.1 M, 10% (v/v) DMS0
pH range: 6-8
Temperature: 30-37xc2x0 C.
Reaction time: 2-20 hours
Substrate concentration: from 0.1 to 0.8 molar
The substrate preferably used is styrene oxide, 3-phenylglycidate, hexane-1, 2-oxide, decane-1, 2-oxide and/or indene oxide in enantiomerically pure form or as enantiomeric mixture.
To develop the color reaction, the pH is then adjusted by adding a base, for example triethylamine. NBP is then added, for example at a concentration in the range from about 3 to 10%, preferably about 5% (w/v) in methoxyethanol. As solubilizer for the pigment formed, for example, triethylene glycol dimethyl ether is added in sufficient quantity. The solution is finally incubated at from about 35 to 45xc2x0 C., preferably 39xc2x0 C., and the absorption is then determined at 560 nm, preferably against a reference at 650 nm. From comparison with an enzyme-free control solution, the decrease in absorption and thus the decrease in epoxide can be determined quantitatively.
The invention also relates to screening methods for detecting microorganisms having epoxide hydrolase activity and/or having the ability to hydrolyze epoxides enantioselectively, comprising the above described detection method. This is particularly suitable for systematic study of strain collections or mutant banks, generated by xe2x80x9cdirected evolutionxe2x80x9d, for epoxide hydrolase activity.
We have found that the above third object was achieved, surprisingly, by providing a process for separating epoxide enantiomer mixtures which comprises
a) incubating an epoxide enantiomer mixture, which comprises an epoxide hydrolase substrate, with an inventive epoxide hydrolase, a microorganism of the genus Streptomyces, an epoxide-hydrolase-containing homogenate thereof or a fraction of this homogenate under reaction conditions;
b) reacting the enantiomer mixture, preferably to achieve a 50% conversion rate; and
c) separating the enantiomer remaining in the reaction mixture from the conversion product and purifying the essentially enantiomerically pure reaction product and/or the essentially enantiomerically pure starting material remaining.
Preferably, enantiomer mixtures of one of the following epoxides is converted: styrene oxide, 3-phenyl-glycidate, hexane-1,2-oxide, decane-1,2-oxide and indene oxide or substituted analogs of these oxides in accordance with the above definition.
The invention further relates to a process for producing epoxide hydrolases (E.C. 3.3.2.3), which comprises
a) producing a cell homogenate from a culture of a microorganism of the genus Streptomyces; 
b) fractionating the homogenate, the resultant fractions being tested for epoxide hydrolase activity, preferably using a detection method based on the color reaction of unreacted epoxide with NBP according to the above definition; and
c) combining fractions having epoxide hydrolase activity and if appropriate further fractionating.
The invention finally relates to the use of an inventive epoxide hydrolase, a microorganism of the genus Streptomyces, an epoxidehydrolase-containing homogenate thereof or a fraction of this homogenate for the enantioselective hydrolysis of epoxides.